It is known that one of the current objectives of pharmaceutical research is to direct drugs to the target organs and/or cells in order to enhance therapeutic efficacy while reducing side effects. One of the means of achieving this objective consists of binding the drug to a molecule, a macromolecule for example, able to play the role of carrier (or "vector") of the drug and bring it specifically to the target organ or target cells.
A number of macromolecules or molecule combinations such as liposomes, nanocapsules, DNA, lectins, antibodies, and lipoproteins have already been used or proposed as biologically active carrier substances.
It is known that lipoproteins constitute an important group of serum proteins comprising a lipid core surrounded by an envelope containing in particular phospholipids and specific proteins (apolipoproteins).
Lipoproteins are the systems that store and carry lipids, particularly cholesterol.
It is known that lipoproteins are classified by density. A distinction is made between:
chylomicrons which are particles with densities less than 0.96; PA1 very-low-density lipoproteins (VLDL) with densities between 0.96 and 1.006 g/cm.sup.3 PA1 low-density lipoproteins (LDL) with densities between approximately 1.019 and 1.063; PA1 high-density lipoproteins (HDL) with densities between approximately 1.063 and 1.21.
Lipoproteins have marked tropism for various cells that have apolipoprotein receptors; in addition, in certain cases, they may be captured by cells in the reticuloendothelial system (phagocytic cells).
Hence it can be seen that lipoproteins are capable of being useful drug carriers. It has already been proposed to use lipoproteins, LDLs in particular, as drug vectors (see, for example, International Patent Application PCT No. WO 86/07540 and European Patent Application No. 0277849).
The low-density lipoproteins (LDLs), which have been the most studied as drug vectors, are spherical particles with diameters of approximately 20 nm. Each particle has a core composed of about 1500 cholesterol ester molecules and an envelope composed of cholesterol, phospholipids, and apolipoproteins (apolipoprotein B or apo B).
Mammal cells have receptors that recognize apo B and bind the LDLs to their surfaces. After binding to the membrane surface of the cells, the LDLs are internalized by endocytosis and carried to the lysosomes where they are broken down to meet the needs of the cell, particularly its cholesterol needs.
If native LDLs, i.e. LDLs that are not chemically modified, are used, the LDL-drug complex behaves like the native LDLs, namely it remains in the bloodstream for two to three days and is recognized by cells that have the apo B receptor (S. M. Brown and J. L. Goldstein, Science, 232, 34-47, 1986). This is the case for cancer cells which have a high level of apo B receptor.
It is also known that chemically modified lipoproteins (acetylates, acetoacetylates, oxide compounds, lactosylates, etc.) are recognized and captured by cells in the reticuloendothelial system, particularly by macrophages. Here again, the (chemically modified) lipoprotein-drug complex behaves in the organism like the original modified lipoprotein and is captured by macrophages through the intermediary of the acetylated LDL receptor (scavenger receptor) (see, for example, J. M. Shaw et al., Proc. Natl. Acad. Sci. USA Vol. 85, pp. 6112-6116 (1988) and M. K. Bijsterbock et al., Molecular Pharmacology, 36, pp. 484-486 (1989)).
In the organism, the transfer of triglycerides and of cholesterol esters and phospholipid between the various classes of lipoproteins (LDLs, VLDLs, HDLs, and chylomicrons) is carried out by proteins known as transfer proteins which have a higher density than lipoproteins, and which are accordingly contained in the residue of ultracentrifuged human or animal plasma after separation of the lipoproteins. Such a centrifugation residue constitutes the fraction known as LPDS (lipoproteindeficient serum), which has in particular the properties of the transfer protein it contains. Some of these transfer proteins have already been isolated and described (see, for example, A. S. Jarnagin et al., Proc. Natl. Acad. Sci. U.S.A. Vol. 84, pp. 1854-57 (1987); Kato et al., J. Biol. Chem. (US), 264, No. 7, 4082-4087 (1989); and Bastiras and Calvert, J. Chromatogr. Biomed. Appl. 383, No. 1, pp. 27-34 (1986)).
Transfer proteins are capable of providing, for example, transport of triglycerides from the VLDLs to the LDLs. They are also capable of transporting triglycerides from artificial triglyceride emulsions to LDLs (see, for example, E. Granot et al., Biochimica et Biophysica Acta 833, pp. 308-315 (1985)).
It has now been discovered that the proteins contained in LPDS are also capable of transporting lipophilic biologically active substances in vitro, other than triglycerides and cholesterol esters, particularly nonphysiologic drugs, from lipid emulsions containing such lipophilic substances to lipoproteins, with these substances being incorporated into the lipoproteins.
This process of incorporation of biologically active substances into lipoproteins is useful because it avoids the use of undesirable substances such as detergents, and thanks to the use of natural transfer agents, it avoids any alteration of LDL biological activity.